Este trabalho foi em parte suportado por concessões para WD de National Institutes of Health (RO1 NS059043 e RO1 ES015988), Multiple Sclerosis Society Nacional, Fundação Roche para Anemia Research, Medical Foundation Feldstein, e os Hospitais Shriners para Crianças. Gostaríamos de agradecer ao Dr. David Prazer e Jennifer Plane para sugestões. Declaramos a inexistência de interesses concorrentes relacionados para este artigo.

Procedimentos detalhados de geração de células precursoras de oligodendrócitos (OPCs) de células de camundongo GFP-Olig2-tronco embrionárias (mESCs): <ol><li> Rato linha celular ES GFP-Olig2 (G-Olig2), é comprado a partir do American Type Culture Collection (ATCC). O mESCs são rotineiramente várias passagens a cada 3 dias para uma camada de fibroblastos irradiados embrionárias de rato alimentador (MEF). As células MEF são banhados em 0,1% de gelatina revestido de seis bem placa de cultura de tecidos, pelo menos, um dia antes passaging CES. O meio de cultura MESC é meio modificado Dulbecco Águia (DMEM) (GIBCO) suplementado com 20% de soro fetal bovino (GIBCO), 2 mM L-glutamina, um piruvato de sódio mM, 0,1 mM β-mercaptoetanol /, 1% aminoácidos não essenciais ( NEAA), e 1.000 U / ml fator inibitório da leucemia. </li><li> colônias MES são tripsinizados (TrypLE, 5 min, 37 ° C) em células individuais e suspensas em knockout soro substituição médio (KSR), então as células são transferidas para um anexo Costar ultra-baixo de seis bem placa (Corning) em uma célula densidade de 50.000 células / cm 2. Nessas condições, mESCs formar corpos embrióides (EBs). Médio KSR consiste de-minimal meio essencial (-MEM) suplementado com 20% KSR, piruvato de sódio 1 mM, NEAA 1% e 0,1 mM β-mercaptoetanol /. </li><li> Do dia 4 ao dia 7, ácido retinóico (RA, 0,2 M) e purmorphamine (Pur, 1 M) são incluídos como mostrado na figura. 1A em KSR ou médio N2. O meio é N2-MEM contendo 1X suplemento N2, piruvato de sódio 1 mM, NEAA 1% e 0,1 mM β-mercaptoetanol /. O meio é alterado todos os dias. </li><li> No dia 8, EBs são desagregados utilizando TrypLE (5min, 37 ° C) e semeadas em 0,01% polyornithine revestido pratos em OPC meio que consiste em N2 médio e fator de crescimento de fibroblastos-2 (FGF-2, 20 ng / ml). O meio é trocada a cada dois dias. </li><li> As células são tripsinizados (TrypLE, 3 min, 37 ° C) e replated aproximadamente a cada semana, quando eles se tornam confluentes. No dia 30, mais de 80% das células mostram GFP/Olig2 expressão e são NG2-positivos, característica de OPCs. </li><li> Para gerar OPCs spiking, o Na + BacMam canal kit é usado para introduzir Na v 1.2 subunidade nestes OPCs MESC derivados. O reagente BacMam (1 ml componente, A) é misturado com PBS para um volume final de 5 ml. Então, o reagente BacMam mista é adicionado em MESC derivados cultura OPCs em um prato de cultura 60 mm (na confluência 50-70%) antes da lavagem com PBS. Após a incubação 2 hr, o reagente BacMam misto é removido e substituído por OPC meio suplementado com 1X enhancer. O enhancer é o componente B reconstituída em DMSO (componente C). Em seguida, após incubação 2 hr adicional de meio com enhancer é removido e substituído com o meio OPC completa. As células são analisadas 24 horas mais tarde. </li></ol> Resultados representativos: Seguindo o protocolo de diferenciação mostrado na figura. 1A, G-Olig2 mESCs foram inicialmente suspensos em meio KSR e para 4 dias para formar corpos embrióides (EBs) (Figura 1B). Então, tratadas seqüencialmente os EBs com AR e Pur. O EBS não apresentaram fluorescência da GFP no dia 4 e expressa de fluorescência da GFP forte em D8 (Figura 1B). Neste ponto do tempo, os EBs foram tripsinizados e banhado em meio suplementado com N2 fatores de crescimento FGF-2. Após o cultivo mais de 22 dias (D30), a porcentagem de células GFP + atingiu 80,3 ± 0,6%, de acordo com nossos resultados de citometria de fluxo. Como mostrado na figura. 1C, GFP + células sobrepostas com NG2 coloração consistente (96,4 ± 1,3% de células GFP positivas foram NG2 positivo, e 94,8 ± 1,5% de células positivas foram NG2 GFP positivas). Assim, estas células expressam GFP/Olig2 e NG2, definindo-os como OPCs. O OPCs MESC derivados (76 células) falhou ao fogo potenciais de ação sobre a despolarização (Fig. 2A). Após transdução com um baculovírus carregando a subunidade Na v 1.2, estes OPCs MESC derivados (94,1%, 16, de 17 células) poderiam ser classificados como spiking (Fig. 2B). <img alt="Figura 1" src="/files/ftp_upload/1960/1960fig1.jpg" /> Figura 1. Diferenciação de GOlig-MESC em OPCs. Scheme (A) mostrando o protocolo do corpo embrióides (EB) e baseada em pequena molécula de diferenciação-driven. No D8, os EBs foram desagregados e banhados. As células foram várias passagens uma vez por semana, quando eles se tornaram confluentes. (B) D8, mas não D4 EBs, mostraram expressão GFP. (C) A imunocoloração de NG2 (vermelho) foi consistente com a GFP (verde) expressão em D30. DAPI (azul) foi utilizado para identificar os núcleos. Barra de escala: 20 mm. <img alt="Figura 2" src="/files/ftp_upload/1960/1960fig2.jpg" /> Figura 2. Geração de OPCs spiking de nonspiking MESC derivados OPCs por vírus mediada Na expressão v canal. (A) O potencial de membrana foi realizada a -60 mV e MESC derivados OPCs não conseguiu acionar os potenciais de ação, mesmo quando a célula foi despolarizada a 0 mV. (B) Um exemplo de MESC derivados OPCdisparo do potencial de ação (destaque em vermelho) após a transdução viral.

<ol>
	<li>Xian, H., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dividing+Olig2-expressing+progenitor+cells+derived+from+ES+cells.">Dividing Olig2-expressing progenitor cells derived from ES cells.</a> Glia. 47 (1), 88-101 (2004).</li><li>Clair, A., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subset+of+ES-cell-derived+neural+cells+marked+by+gene+targeting.">A subset of ES-cell-derived neural cells marked by gene targeting.</a> Stem Cells. 21 (1), 41-49 (2003).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154-156 (1981).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Shin, S., Xue, H., Mattson, M. P., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-dependent+Olig2+expression+in+motor+neurons+and+oligodendrocytes+differentiated+from+embryonic+stem+cells.">Stage-dependent Olig2 expression in motor neurons and oligodendrocytes differentiated from embryonic stem cells.</a> Stem Cells Dev. 16, 131-141 (2007).</li><li>Billon, N., Jolicoeur, C., Ying, Q. L., Smith, A., Raff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+timing+of+oligodendrocyte+development+from+genetically+engineered,+lineage-selectable+mouse+ES+cells.">Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells.</a> J Cell Sci. 115, 3657-3665 (2002).</li><li>Nishiyama, A., Lin, X. H., Giese, N., Heldin, C. H., Stallcup, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-localization+of+NG2+proteoglycan+and+PDGF+alpha-receptor+on+O2A+progenitor+cells+in+the+developing+rat+brain.">Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain.</a> J Neurosci Res. 43, 299-314 (1996).</li><li>Ligon, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+NG2+neural+progenitor+cells+requires+Olig+gene+function.">Development of NG2 neural progenitor cells requires Olig gene function.</a> Proc Natl Acad Sci U S A. 103, 7853-7858 (2006).</li><li>Dawson, M. R., Polito, A., Levine, J. M., Reynolds, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-expressing+glial+progenitor+cells:+an+abundant+and+widespread+population+of+cycling+cells+in+the+adult+rat+CNS.">NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS.</a> Mol Cell Neurosci. 24, 476-488 (2003).</li><li>Chittajallu, R., Aguirre, A., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-positive+cells+in+the+mouse+white+and+grey+matter+display+distinct+physiological+properties.">NG2-positive cells in the mouse white and grey matter display distinct physiological properties.</a> J Physiol. 561, 109-122 (2004).</li><li>Karadottir, R., Hamilton, N. B., Bakiri, Y., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiking+and+nonspiking+classes+of+oligodendrocyte+precursor+glia+in+CNS+white+matter.">Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter.</a> Nat Neurosci. 11, 450-456 (2008).</li></ol>

Não há conflitos de interesse declarados.

Células-tronco embrionárias (CES), isolado a partir de um embrião de blastocisto, podem se diferenciar em todas as linhagens de células do organismo 3, 4, provendo uma in vitro sistema modelo para estudar o desenvolvimento de mamíferos adiantados, incluindo especificação de oligodendrócitos. mESCs foram mostrados para se diferenciar em células precursoras de oligodendrócitos (OPCs) com o tratamento de sonic hedgehog (Shh) 5. Além disso, o Shh induzida diferenciação OPC a partir mESCs mantém o timing correto observado no desenvolvimento embrionário 6. Assim, a natureza da diferenciação in vitro de mESCs OPC é considerado para ser coerente com o que foi aprendido com desenvolvimento in vivo. Aqui, usando a pequenas moléculas RA eo Pur agonista Shh, conseguimos diferenciada do mESCs G-Olig2 em GFP + + Olig2 NG2 + OPCs com alta eficiência. OPCs, caracterizado pela expressão do proteoglicano NG2 7 e do fator de transcrição hélice-alça-hélice Olig2 8, gerar oligodendrócitos no SNC em desenvolvimento e maduras, onde constituem uma percentagem significativa (~ 5%) do total de células e são os principal tipo de célula em proliferação 9. Por quase duas décadas pesquisadores demonstraram Na canais V são expressos em uma subpopulação de OPCs e pode ser activado a despolarização 10, 11. Além disso, uma observação recente impressionante foi que em 9 no SNC de ratos situ substância branca, OPCs (~ 50%) gerados potenciais de ação quando despolarizada, dependendo da expressão de voltagem-dependentes de sódio (Na V) canais, e, portanto, poderia ser subdividida em spiking e nonspiking subpopulações. No entanto, as funções dessas propriedades spiking são ainda desconhecidos. Descobrimos que, eletrofisiologicamente, o MESC OPCs derivados diferenciada com o protocolo Shh-dependente, não eram o mesmo que o cérebro em OPCs situ. Depois de introduzir subunidade Na V 1.2, estas silêncio MESC derivados OPCs eram capazes de spiking. Assim, o spiking / nonspiking MESC derivados OPCs e protocolo de diferenciação descrito aqui pode facilitar (1) estudar as diferenças funcionais entre spiking e nonspiking OPCs, (2) fatores de triagem novo que poderia promover a expressão dos canais de sódio na OPCs MESC derivados, ( 3) desenvolver e optimizar o protocolo de diferenciação de OPCs de humano ESC ou células-tronco pluripotentes induzidas (iPSCs).

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<td>MEFs</td>
<td>&nbsp;</td>
<td>GlobalStem </td>
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<td>TrypLE Express</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12604</td>
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<td>Retinoic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R-2625</td>
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<td>Purmorphamine</td>
<td>&nbsp;</td>
<td>Cayman Chemical</td>
<td>10009634</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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differentiation of embryonic stem cells into oligodendrocyte precursors

Oligodendrócitos são as células myelinating do sistema nervoso central. Para terapia celular regenerativa em doenças desmielinizantes, há um interesse significativo na derivação de uma população pura de linhagem comprometida células precursoras de oligodendrócitos (OPCs) para transplante. OPCs são caracterizadas pela atividade da Olig2 fator de transcrição e expressão de superfície de um proteoglicano NG2. Usando o GFP-Olig2 (G-Olig2) mouse com células-tronco embrionárias (MESC), linha repórter, nós otimizamos as condições para a diferenciação de mESCs em GFP + + Olig2 NG2 + OPCs. Em nosso protocolo, primeiro descrever a geração de corpos embrióides (EBs) de mESCs. Segunda, descrevemos o tratamento de MESC derivados EBs com pequenas moléculas: (1) ácido retinóico (AR) e (2) um ouriço sónico purmorphamine agonista (Shh) (Pur), sob condições de cultura definidas para direcionar a diferenciação EB na linhagem oligodendroglial. Por esta abordagem, OPCs pode ser obtido com alta eficiência (&gt; 80%) em um período de 30 dias. Células derivadas de mESCs neste protocolo são fenotipicamente semelhante a OPCs derivados de cultura de tecidos primários. O OPCs MESC derivados não apresentam a propriedade spiking descrito para uma subpopulação de OPCs cérebro in situ. Para estudar esta propriedade eletrofisiológicos, descrevemos a geração de spiking MESC derivados OPCs por ectopicamente expressando Na<sub&gt; V</sub&gt; 1,2 subunidade. As células spiking e nonspiking obtidos a partir deste protocolo vai ajudar a avançar os estudos funcionais sobre as duas subpopulações de OPCs.

Watch this Scientific Journal Video about Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors at JoVE.com

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Descrevemos uma molécula baseada em pequenas protocolo para a diferenciação de células-tronco embrionárias de camundongos em células precursoras de oligodendrócitos (OPCs). Este protocolo gera Olig2 + + NG2 OPCs com alta eficiência por 30 dias de diferenciação. Nós também descrevem um método para gerar &quot;spiking&quot; OPCs que pode disparar potenciais de ação.

Diferenciação de células-tronco embrionárias em precursores oligodendroglial

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

differentiation-embryonic-stem-cells-into-oligodendrocyte

Research

JoVE Journal

Biology

15.2K visualizações.  School of Medicine, University of California, Davis. Descrevemos uma molécula baseada em pequenas protocolo para a diferenciação de células-tronco embrionárias de camundongos em células precursoras de oligodendrócitos (OPCs). Este protocolo gera Olig2 + + NG2 OPCs com alta eficiência por 30 dias de diferenciação. Nós também descrevem um método para gerar "spiking" OPCs que pode disparar potenciais de ação.

Vídeo: Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagação de-tronco embrionárias humanas (ES) Células

Biologia

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Cultura de rato precursores das células-tronco neurais

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivação de Células-Tronco Hematopoéticas murino de Células-Tronco Embrionárias

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivação de células estaminais embrionárias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Cromatina imunoprecipitação de células estaminais embrionárias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Na diferenciação in vitro de rato-tronco embrionárias (MES) células usando o método da gota de suspensão

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolamento e derivação de células embrionárias de rato Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivação eficiente dos recursos humanos Precursores cardíaca e Cardiomyocytes pluripotentes a partir de células estaminais embrionárias humanas com a indução de moléculas pequenas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Diferenciação eficiente de células-tronco embrionárias em neurônios motores

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Diferenciação de camundongos neonatos pele derivadas de células-tronco em células germinativas semelhantes<em&gt; In vitro</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...